colorimeter

it is presentation of instrument of meical laboratory

1. COLORIMETER By Apurva J ha

2. Colorimeter is instrument which is used in the measurement of the luminious intensity of light. Invented by Louis Jules Duboscq in 1870. INTRODUCTION

3. colorimeter

4. Colorimetry is the technique frequently used in biochemical investigations,involves the quantitative estimation of colors. Color can be produced by any substance when it binds with color forming chromogens . The difference in color intensity results in the difference in the absorption of light. The intensity of color is directly proportional to the concentration of the compound being measured PRINCIPLE

5. Wavelength between 400nm to 700nm form the visible spectrum of light visible band of light in electromagnetic spectrum Relationship between wavelength & colour

6. Wavelength (nm) Spectrum region Colour absorbed Colour transmitted 400-420 Visible Violet Green-yellow 420-500 Visible Blue yellow 500-570 Visible Green Red 570-600 Visible yellow Blue 600-630 Visible orange Green-blue 630-700 Visible Red Green

7. Light falling on a color solution is either absorbed, reflected or transmitted. I o= I t + I a Absorption & transmittance of light I o I t I a

8. Transmittance (T) It is ratio of the intensity of the transmitted light over the intensity of the incident light. P ercentage transmission(%T) = I t /I i X100 Absorbance Absorbance is the amount of light absorbed by a sample.  It is calculated from T or %T using the following equations. Relationship between absorbance and transmittance

9. A = O.D = Log 1/T = Log( I00/ %T) = Log100-Log%T i.e. O.D. = 2-Log (%T)

10. The nature of light absorbing substance . Wavelength of light and Amount of light absorbing substance in the light path, which in turn depends on the concentration of light absorbing substance and depth of the solution through which light passes. . Transmittance of a solution containing light absorbing substance depends upon

11. Absorbance Concentration Concentration % transmission (a) Relation between absorbance & concentration. (b) Percentage transmission & concentration.

12. The relationship between concentration of the compound and color intensity is given by Beer’s law and Lamberts law

13. Beer’s law When monochromatic light passes through a light absorbing medium, the intensity of the transmitted light decreases exponentially as the concentration of the light absorbing material increases. A α C Where A is light absorbed and C is concentration of the solution. Basis of colorimetric techniques

14. When monochromatic light passes through a coloured solution, the amount of light absorbed increases with the increase in thickness of the layer of the solution through which the light passes. A α L Where L = length of light path Lamberts law

15. By combining above equations,we get A α CL A = KCL Where k=constant for coloured solution

16. For standard solution : As =Ks Cs Ls For unknown solution : Au =Ku Cu Lu Au =absorbance of unknown solution C u = conc of unknown solution AS =absorbance of std solution CS = conc of std solution But Ks =Ku & Ls =Lu Au/As = Cu/Cs Cu= Au/As X Cs

17. Light source Monochromator / wavelength selector F ilter Solution/sample holder Cuvette Photosensitive detector system Measuring device Parts of colorimeter

18. C olorimeter Wavelength selection, Printer button Concentration factor adjustment, UV mode selector (Deuterium lamp) Readout Sample compartment Zero control (100% T), Sensitivity switch

19. Flow representation of colorimeter

20. Common source is a tungsten-filament lamp, higher powered tungsten –halogen (quartz-iodine) lamp. Factor of light source are range, spectral distribution, stability of radiant energy and temperature.. Light source

21. Mono = single. Chromatic- colour Monochromatic light is the single colour band of light. Monochromator and filters are used to split the light from the light source. Simple filters are either coloured glass or suitably dyed gelatin sandwiched in a glass. filters range is 400-680 nm Monochromator /wavelength selector

22. Complementary filters for coloured solutions . The selected filters has the color to the complementary to that of the color of unknown solution

23. Color Wheel ( R O Y G B I V ) Compl e mentary colors lie across the diameter on the color wheel and combine to form “white light” , so the color of a compound seen by the eye is the complement of the color of light absorbed by a colored compound; thus it completes the color.

24. ג max It is maximum absorbance by the solution at one particular wavelength .

25. Cuvette are rectangular cell , square cell or circular one Made up of optical glass for visible wavelength. Common one is square,rectangular to avoid refraction artefacts . dimension of cuvette is 1cm. Solution holder

26. cuvettes

27. Photo sensitive detector when light falls on these electric elements electric current is generated which deflects a galvanometer needle. The meter reading is proportional to the light intensity ,these photosensitive detectors are also referred to as photoelectric cells. One of the common used photo cell is Barrier layer cell.

28. Measuring device Current from detector is fed to a sensitive suitable measuring device, usually galvanometer . Absorbance scale ranges from 0 to 2 ,while % transmission scale ranges from 0 to 100. Zero absorbance = 100% transmission Infinite absorbance =0 transmission.

29. Power required 230 V AC ± 10% 50 Hz, 10 VA Filters Separate glass filters 400, 420, 470, 500, 530, 620, 660 & 700 nm Readout 2½ digit LED display Measurement a) Transmittance 'T'-0-100% b) Absorption Log 'T'-0-2 Light source LED of infinite life Detector Photo cell Test tube 12 mm Dia. with 1ml mark and position mark Warming time 5 minutes Weight / Body 1 Kg. (approx.) / ABS Dimension 95 mm (H) x 225 mm (W) x 215 mm (L) Sample quantity 1ml Accessories 5 matched test tubes, Dust proof Some features of EQ 650 A Reads ‘%T’ and ‘OD’

30. Advantage It is inexpensive . Very well applicable for quantitative analysis of colored compounds. Easily carriable and transportable. COLORIMETER

31. Disadvantage Cannot be used for colorless compounds. It does not work in UV and IR regions. We cannot set specific wavelength, as we have to set a range as a parameter . Similar colors from interfering substances can produce errors in results   . COLORIMETER

32. It is widely used in hospital & laboratory for estimation of biochemical samples , like plasma, serum, cerebrospinal fluid ( csf ) , urine. It is also used to quantitative estimation of serum components as well as glucose, proteins and other various biochemical compound. T hey are used by the food industry and by manufacturers of paints and textiles . Application

33. T hey are used to test for water quality, by screening for chemicals such as chlorine, fluoride, cyanide, dissolved oxygen, iron, molybdenum, zinc and hydrazine. They are also used to determine the concentrations of plant nutrients (such as phosphorus, nitrate and ammonia) in the soil or hemoglobin in the blood and to identify substandard and counterfeit drugs . 

34. R eferences Clinical chemistry – michael l.bishop A book of medical science- J.ochei Practical biochemistry- keith wilson & john walker Clinical chemistry &molecular diagnostics- Tietz

35. Water blank Reagent blank Standard solution Use of blank

36. THANK YOU